Methods for the treatment of thrombosis

ABSTRACT

A fibrinolytically active metalloproteinase polypeptide (called “novel acting thrombolytic”) which is useful for blood clot lysis in vivo and methods and materials for its production by recombinant expression are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/441,667, filed May 20, 2003, which is a divisional application ofU.S. Ser. No. 09/846,729, filed May 1, 2001, now U.S. Pat. No.6,617,145, which is a divisional application of U.S. Ser. No.09/411,329, filed Oct. 1, 1999, now U.S. Pat. No. 6,261,820, from whichapplications priority is claimed pursuant to 35 U.S.C. §120, and whichapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to a fibrinolytically active metalloproteinase ofnon-naturally occurring sequence, to combinant methods for itsmanufacture, and to its use in treating thrombosis in vivo.

BACKGROUND OF THE INVENTION

Fibrolase is an enzymatically active polypeptide (specifically, ametalloproteinase) composed of 203 amino acid residues that wasoriginally isolated by purification from the venom of the SouthernCopperhead snake; U.S. Pat. No. 4,610,879, issued Sep. 9, 1986 (Marklandet al.); and Guan et al., Archives of Biochemistry and Biophysics,Volume 289, Number 2, pages 197-207 (1991). The enzyme exhibits directfibrinolytic activity with little or no hemorrhagic activity, and itdissolves blood clots made either from fibrinogen or from whole blood.

The amino acid sequence of fibrolase has also been determined, withmethods described for recombinant production in yeast and use for thetreatment of thrombembolic conditions in vivo; Randolph et al., ProteinScience, Cambridge University Press (1992), pages 590-600, and EuropeanPatent Application No. 0 323 722 (Valenzuela et al.), published Jul. 12,1989.

SUMMARY OF THE INVENTION

This invention provides a fibinolytic metalloproteinase having thenon-naturally occurring linear array of amino acids depicted in SEQ IDNO: 1, also referred to herein as “novel acting thrombolytic” (or“NAT”). Also provided are nucleic acid molecules, such as the one of SEQID NO: 2 and variants thereof encoding NAT.

The term “mature” is used in its conventional sense to refer to thebiologically active polypeptide which has been enzymatically processedin situ in the host cell to cleave it from the prepro region.

Because of its fibrinolytic activity, NAT is useful in vivo as a bloodclot lysing agent to treat thrombosis in a mammal (including rats, pigsand humans).

The NAT polypeptide of this invention provides advantages over naturallyoccurring fibrolase as a therapeutic agent (i.e., the fibrinolyticpolypeptide found in snake venom). Native fibrolase is known to containseveral alternate N-termini: QQRFP, EQRFP and ERFP (in which “E”designates a cyclized glutamine, or pyroglutamic acid). Morespecifically, starting with an N-terminus composed of QQRFP, thefibrolase molecule undergoes degradation to result in two isoforms,having N-terminal sequences of EQRFP and ERFP, respectively. Recombinantfibrolase as produced in yeast typically yields a mixture of all threeof these forms and is thus not homogeneous. See Loayza et al., Journalof Chromatography, B 662, pages 227-243 (1994). Moreover, the cyclizedglutamine residue results in a “blocked” N-terminus which makessequencing impossible.

In contrast, the recombinant NAT of this invention provides a singlespecies: only one N-terminus is typically produced. The result isgreater homogeneity of the end product compared to recombinantfibrolase, which is beneficial when medical applications are theintended end use.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts in linear fashion the full amino acid sequence of NAT(SEQ ID NO: 3), consisting of the “prepro” region (underscored), whichincludes the “signal” peptide, and the mature polypeptide(non-underscored).

DETAILED DESCRIPTION OF THE INVENTION

NAT may be produced by recombinant expression of the nucleic acidmolecule of SEQ ID NO: 4, which encodes the full amino acid sequence ofNAT (SEQ ID NO: 3), including the prepro region from nucleotides 1-783and mature polypeptide from nucleotides 784-1386, in a suitable host.Following expression, the prepro region is enzymatically processed offin the host cell to yield the mature active polypeptide (SEQ ID NO: 1).

Preferably, NAT is produced recombinantly in yeast, as will be explainedin greater detail further below.

The mature polypeptide (SEQ ID NO: 1) which is thus produced may or maynot have an amino terminal methionine, depending on the manner in whichit is prepared. Typically, an amino terminal methionine residue will bepresent when the polypeptide is produced recombinantly in anon-secreting bacterial (e.g., E. coli) strain as the host.

Besides the nucleic acid molecule of SEQ ID NO: 2, also utilizable aredegenerate sequences thereof which encode the same polypeptide. Thepresent invention also embraces nucleic acid molecules that may encodeadditional amino acid residues flanking the 5′ or 3′ portions of theregion encoding the mature polypeptide, such as sequences encodingalternative pre/pro regions (i.e., sequences responsible for secretionof the polypeptide through cell membranes) in place of the “native”pre/pro region (i.e., found in naturally occurring fibrolase). Theadditional sequences may also be noncoding sequences, includingregulatory sequences such as promoters of transcription or translation,depending on the host cell.

NAT can be prepared using well known recombinant DNA technology methods,such as those set forth in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and/or Ausubel et al., editors, Current Protocols inMolecular Biology, Green Publishers Inc. and Wiley and Sons, New York(1994). A DNA molecule encoding the polypeptide or truncated versionthereof may be obtained, for example, by screening a genomic or cDNAlibrary, or by PCR amplification, to obtain a nucleic acid moleculeencoding fibrolase, followed by replacement of the codons encoding theN-terminal amino acid residues QQR with a codon for serine (S).Alternatively, a DNA molecule encoding NAT may be prepared by chemicalsynthesis using methods well known to the skilled artisan, such as thosedescribed by Engels et al. in Angew. Chem. Intl. Ed., Volume 28, pages716-734 (1989). Typically, the DNA will be several hundred nucleotidesin length. Nucleic acids larger than about one hundred nucleotides canbe synthesized as several fragments using these same methods and thefragments can then be ligated together to form a nucleotide sequence ofthe desired length.

The DNA molecule is inserted into an appropriate expression vector forexpression in a suitable host cell. The vector is selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery, such that expression of the DNAcan occur). The polypeptide may be expressed in prokaryotic, yeast,insect (baculovirus systems) or eukaryotic host cells, although yeast ispreferred as will be explained in greater detail further below.

The vectors used in any of the host cells to express NAT may alsocontain a 5′ flanking sequence (also referred to as a “promoter”) andother expression regulatory elements operatively linked to the DNA to beexpressed, as well as enhancer(s), an origin of replication element, atranscriptional termination element, a complete intron sequencecontaining a donor and acceptor splice site, a signal peptide sequence,a ribosome binding site element, a polyadenylation sequence, apolylinker region for inserting the nucleic acid encoding NAT, and aselectable marker element. Each of these elements is discussed below.

Optionally, the vector may also contain a “tag” sequence, i.e., anoligonucleotide sequence located at the 5′ or 3′ end of thepolypeptide-coding sequence that encodes polyHis (such as hexaHis) oranother small immunogenic sequence (such as the c-myc or hemagglutininepitope, for which antibodies, including monoclonal antibodies, arecommercially available). This tag will be expressed along with NAT, andcan serve as an affinity tag for purification of this polypeptide fromthe host cell. Optionally, the tag can subsequently be removed from thepurified polypeptide by various means, for example, with use of aselective peptidase.

The 5′ flanking sequence may be the native 5′ flanking sequence, or itmay be homologous (i.e., from the same species and/or strain as the hostcell), heterologous (i.e., from a species other than the host cellspecies or strain), hybrid (i.e., a combination of 5′ flanking sequencesfrom more than one source), or synthetic. The source of the 5′ flankingsequence may be any unicellular prokaryotic or eukaryotic organism, anyvertebrate or invertebrate organism, or any plant, provided that the 5′flanking sequence is functional in, and can be activated by the hostcell machinery.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofNAT. If the vector of choice does not contain an origin of replicationsite, one may be chemically synthesized based on a known sequence, andthen ligated into the vector.

The transcription termination element is typically located 3′ to the endof the polypeptide coding sequence and serves to terminate transcriptionof the mRNA. Usually, the transcription termination element inprokaryotic cells is a G-C rich fragment followed by a poly T sequence.While the element is easily cloned from a library or even purchasedcommercially as part of a vector, it can also be readily synthesizedusing known methods for nucleic acid synthesis.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that: (a) conferresistance to antibiotics or other toxins, for example, ampicillin,tetracycline or kanamycin for prokaryotic host cells, zeocin for yeasthost cells, and neomycin for mammalian host cells; (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers for usein prokaryotic expression are the kanamycin resistance gene, theampicillin resistance gene, and the tetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (for prokaryotes) or the Kozak sequence (for eukaryotes), isnecessary for the initiation of translation of mRNA. The element istypically located 3′ to the promoter and 5′ to the coding sequence ofthe polypeptide to be synthesized. The Shine-Dalgarno sequence is variedbut is typically a polypurine (i.e., having a high A-G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector. The Kozak sequence typically includes sequencesimmediately before and after the initiating codon. A preferred Kozaksequence is one that is associated with a high efficiency of initiationof translation at the AUG start codon.

In those cases where it is desirable for NAT polypeptide to be secretedfrom the host cell, a signal sequence may be used to direct thepolypeptide out of the host cell where it is synthesized. Typically, thesignal sequence is positioned in the coding region of nucleic acidsequence, or directly at the 5′ end of the coding region. Many signalsequences have been identified, and any of them that are functional inthe selected host cell may be used here. Consequently, the signalsequence may be homologous or heterologous to the polypeptide.Additionally, the signal sequence may be chemically synthesized usingmethods referred to above.

After the vector has been constructed and a nucleic acid has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or polypeptideexpression.

As mentioned, host cells may be prokaryotic (such as E. coli) oreukaryotic (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, whether it be yeast or some other host, when culturedunder appropriate conditions can synthesize NAT, which can subsequentlybe collected from the culture medium (if the host cell secretes it intothe medium) or directly from the host cell producing it (if it is notsecreted). After collection, NAT polypeptide can be purified usingmethods such as molecular sieve chromatography, affinity chromatography,and the like.

Selection of the host cell will depend in large part on whether themanner in which the host cell is able to “fold” NAT into its nativesecondary and tertiary structure (e.g., proper orientation of disulfidebridges, etc.) such that biologically active material is prepared by thecell. However, even where the host cell does not synthesize biologicallyactive material, it may be “folded” after synthesis using appropriatechemical conditions, such as ones that are known to those skilled in theart. In either case, proper folding can be inferred from the fact thatbiologically active material has been obtained.

Suitable host cells or cell lines may be mammalian cells, such asChinese hamster ovary cells (CHO) or 3T3 cells. The selection ofsuitable mammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. Other suitable mammalian cell lines are the monkeyCOS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Still other suitable mammalian cell lines include butare not limited to, HeLa, mouse L-929 cells, 3T3 lines derived fromSwiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

Also useful as host cells are bacterial cells. For example, the variousstrains of E. coli (e.g., HB101, DH5a, DH10, and MC1061) are well-knownas host cells in the field of biotechnology. Various strains of B.subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., andthe like, may also be employed. Additionally, many strains of yeastcells known to those skilled in the art are also available as host cellsfor expression of the polypeptide of the present invention. Also, wheredesired, insect cells may be utilized as host cells. See, for example,Miller et al., Genetic Engineering, Volume 8, pages 277-298 (1986).

Insertion (also referred to as “transformation” or “transfection”) ofthe vector into the selected host cell may be accomplished using suchmethods as calcium phosphate, electroporation, microinjection,lipofection or the DEAE-dextran method. The method selected will in partbe a function of the type of host cell to be used. These methods andother suitable methods are well known to the skilled artisan, and areset forth, for example, in Sambrook et al., above.

The host cells containing the vector may be cultured using standardmedia well known to the skilled artisan. The media will usually containall nutrients necessary for the growth and survival of the cells.Suitable media for culturing E. coli cells are, for example, Luria Broth(LB) and/or Terrific Broth (TB). Suitable media for culturing eukaryoticcells are RPMI 1640, MEM, DMEM, all of which may be supplemented withserum and/or growth factors as required by the particular cell linebeing cultured. A suitable medium for insect cultures is Grace's mediumsupplemented with yeastolate, lactalbumin hydrolysate and/or fetal calfserum, as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of NAT produced in the host cell can be evaluated usingstandard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If NAT is secreted from the host cells other than gram-negativebacteria, the majority will likely be found in the cell culture medium.If NAT is secreted from gram-negative bacteria, it will to some degreebe found in the periplasm. If NAT is not secreted, it will be present inthe cytoplasm.

For intracellular NAT, the host cells are typically first disruptedmechanically. For NAT having a periplasmic location, either mechanicaldisruption or osmotic treatment can be used to release the periplasmiccontents into a buffered solution. NAT polypeptide is then isolated fromthis solution. Purification from solution can thereafter be accomplishedusing a variety of techniques. If NAT has been synthesized so that itcontains a tag such as hexahistidine or other small peptide at eitherits carboxyl or amino terminus, it may essentially be purified in aone-step process by passing the solution through an affinity columnwhere the column matrix has a high affinity for the tag or for thepolypeptide directly (i.e., a monoclonal antibody). For example,polyhistidine binds with great affinity and specificity to nickel, thusan affinity column of nickel (such as the Qiagen nickel columns) can beused for purification. (See, for example, Ausubel et al., editors,Current Protocols in Molecular Biology, above).

Where, on the other hand, the polypeptide has no tag and no antibodiesare available, other well known procedures for purification can be used.Such procedures include, without limitation, ion exchangechromatography, molecular sieve chromatography, reversed phasechromatography, HPLC, native gel electrophoresis in combination with gelelution, and preparative isoelectric focusing (“Isoprime”machine/technique, Hoefer Scientific). In some cases, two or more ofthese techniques may be combined to achieve increased purity.

Especially preferred for use in the production of NAT are yeast cells,and most advantageously those of the yeast genus known as Pichia (e.g.,Pichia pastoris), because of the greater efficiency of refoldingcompared to, for instance, bacterial cells such as E. coli. Suitablerecombinant methods of expression for this yeast strain are described inU.S. Pat. Nos. 4,855,231 (Stroman et al.), 4,812,405 (Lair et al.),4,818,700 (Cregg et al.), 4,885,242 (Cregg) and 4,837,148 (Cregg), thedisclosures of which are incorporated herein by reference.

Notably, Pichia cells can also be used to express fibrolase with similarefficiency from DNA molecules encoding this metalloproteinase, and sucha method constitutes an additional aspect of the present invention.Fibrolase is a known metalloproteinase which has been described in thescientific and patent literature; see Randolph et al., and EuropeanPatent Application No. 0 323 722, cited above. Typically, the fibrolaseto be expressed will be of SEQ ID NO: 5, which is encoded by the cDNAmolecule of SEQ ID NO: 6 (or variants thereof encoding the same aminoacid sequence). The expression of fibrolase in such a system willtypically involve a DNA molecule of SEQ ID NO: 7, which encodes “prepro”sequence (nucleotides 1-783) in addition to the “mature” polypeptide(nucleotides 784-1392).

Chemically modified versions of NAT in which the polypeptide is linkedto a polymer or other molecule to form a derivative in order to modifyproperties are also included within the scope of the present invention.For human therapeutic purposes especially, it may be advantageous toderivatize NAT in such a manner by the attachment of one or more otherchemical moieties to the polypeptide moiety. Such chemical moieties maybe selected from among various water soluble polymers. The polymershould be water soluble so that the NAT polypeptide to which it isattached is miscible in an aqueous environment, such as a physiologicalenvironment. The water soluble polymer may be selected from the groupconsisting of, for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random or non-random copolymers (see further below regarding fusionmolecules), and dextran or poly(n-vinyl pyrolidone)polyethylene glycol,propylene glycol homopolymers, polypropylene oxide/ethylene oxideco-polymers, polyoxyethylated polyols, polystyrenemaleate and polyvinylalcohol. Polyethylene glycol propionaldenhyde may have advantages inmanufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 2 kilodaltons (kDa) and about 100 kDa (the term “about”indicating that in preparations of polyethylene glycol, some moleculeswill weigh more, some less, than the stated molecular weight) for easein handling and manufacturing. Other sizes may be used, depending on thedesired therapeutic profile (e.g., the duration of sustained releasedesired, the effects, if any on biological activity, the ease inhandling, the degree or lack of antigenicity and other known effects ofthe polyethylene glycol on a therapeutic protein).

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the effect on function. One maymono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to NAT polypeptidemolecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted polypeptide or polymer)will be determined by factors such as the desired degree ofderivatization (e.g., mono, di-, tri-, etc.), the molecular weight ofthe polymer selected, whether the polymer is branched or unbranched, andthe reaction conditions.

The chemical moieties should be attached to NAT with consideration ofeffects on functional or antigenic domains of the polypeptide. There area number of attachment methods available to those skilled in the art.See, for example, EP 0 401 384 (coupling PEG to G-CSF), and Malik etal., Experimental Hematology, Volume 20, pages 1028-1035 (1992)(reporting the pegylation of GM-CSF using tresyl chloride). By way ofillustration, polyethylene glycol may be covalently bound through aminoacid residues via a reactive group, such as, a free amino or carboxylgroup. Reactive groups are those to which an activated polyethyleneglycol molecule (or other chemical moiety) may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residue. Those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues, and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecule(s) (orother chemical moiety). Preferred for manufacturing purposes isattachment at an amino group, such as at the N-terminus or to a lysinegroup. Attachment at residues important for receptor binding should beavoided if receptor binding is desired.

One may specifically desire N-terminally chemically modifiedderivatives. Using polyethylene glycol as an illustration, one mayselect from a variety of polyethylene glycol molecules (by molecularweight, branching, etc.), the proportion of polyethylene glycolmolecules to polypeptide molecules in the reaction mixture, the type ofpegylation reaction to be performed, and the method of obtaining theselected N-terminally pegylated NAT. The method of obtaining theN-terminally pegylated preparation (i.e., separating this moiety fromother monopegylated moieties if necessary) may be by purification of theN-terminally pegylated material from a population of pegylated NATmolecules. Selective N-terminal chemical modification may beaccomplished by reductive alkylation which exploits differentialreactivity of different types of primary amino groups (lysine versus theN-terminal) available for derivatization. See PCT application WO96/11953, published Apr. 25, 1996. Under the appropriate reactionconditions, substantially selective derivatization of NAT at theN-terminus with a carbonyl group containing polymer is achieved. Forexample, one may selectively N-terminally pegylate NAT by performing thereaction at a pH which allows one to take advantage of the pK_(a)differences between the ε-amino group of the lysine residues and that ofthe α-amino group of the N-terminal residue of the polypeptide. By suchselective derivatization, attachment of a polymer to a polypeptide iscontrolled: the conjugation with the polymer takes place predominantlyat the N-terminus of the polypeptide and no significant modification ofother reactive groups, such as lysine side chain amino groups, occurs.Using reductive alkylation, the polymer may be of the type describedabove, and should have a single reactive aldehyde for coupling to thepolypeptide. Polyethylene glycol propionaldehyde, containing a singlereactive aldehyde, may be used.

NAT or chemically modified derivatives in accordance with the inventionmay be formulated for in vivo administration, and most preferably viaintra-thrombus (i.e., via localized delivery directly to the site of theclot in the blood vessel, e.g., as by catheter). Systemic delivery isnormally not preferred due to the likelihood that innate a2macroglobulin in the general circulation may complex with NAT to preventinteraction with fibrin or fibrinogen, thus impairing clot lysis.However, there may be instances where larger amounts of NAT can be usedwhich exceed the circulating levels of α2 macroglobulin, thus enablingsystemic administration and delivery. In general, encompassed within theinvention are pharmaceutical compositions comprising effective amountsof NAT together with pharmaceutically acceptable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. By“effective amount” is meant amount sufficient to produce a measurablebiological effect (i.e., a thrombolytically effective amount whicheffects lysis of the blood clot or clots being treated).

Typically, NAT will be in highly purified form, and any pharmaceuticalcomposition being used as the delivery vehicle will normally bepresterilized for use, such as by filtration through sterile filtrationmembranes.

One skilled in the art will be able to ascertain effective dosages byadministration and observing the desired therapeutic effect. Particulareffective doses within this range will depend on the particular disorderor condition being treated, as well as the age and general health of therecipient, and can be determined by standard clinical procedures. Wherepossible, it will be desirable to determine the dose-response curve ofthe pharmaceutical composition first in vitro, as in bioassay systems,and then in useful animal model systems in vivo prior to testing inhumans. The skilled practitioner, considering the therapeutic context,type of disorder under treatment, and other applicable factors, will beable to ascertain proper dosing without undue effort. Typically, apractitioner will administer the NAT composition until a dosage isreached that achieves the desired effect (i.e., lysis of the bloodclot). The composition may be administered as a single dose, or as twoor more doses (which may or may not contain the same amount ofpolypeptide) over time, or on a continuous basis.

NAT may also be used to generate antibodies in accordance with standardmethods. The antibodies may be polyclonal, monoclonal, recombinant,chimeric, single-chain and/or bispecific, etc. To improve the likelihoodof producing an immune response, the amino acid sequence of NAT can beanalyzed to identify portions of the molecule that may be associatedwith increased immunogenicity. For example, the amino acid sequence maybe subjected to computer analysis to identify surface epitopes, such asin accordance with the method of Hope and Woods, Proceedings of theNational Academy of Science USA, Volume 78, pages 3824-3828 (1981).

Various procedures known in the art can be used for the production ofpolyclonal antibodies which recognize epitopes of NAT. For theproduction of antibody, various host animals can be immunized byinjection with the polypeptide, including but not limited to rabbits,mice, rats, etc. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's, mineral gels such as aluminum hydroxide (alum),surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BacilleCalmette-Guerin and Corynebacterium parvum.

For the preparation of monoclonal antibodies directed toward NAT, anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture may be used. For example, the hybridomatechnique originally developed by Kohler and Milstein which is describedin Nature, Volume 256, pages 495-497 (1975), as well as the triomatechnique, the human B-cell hybridoma technique described by Kozbor etal. in Immunology Today, Volume 4, page 72 (1983), and the EBV-hybridomatechnique to produce monoclonal antibodies described by Cole et al. in“Monoclonal Antibodies and Cancer Therapy”, Alan R. Liss, Inc., pages77-96 (1985), are all useful for preparation of monoclonal antibodies inaccordance with this invention.

The antibodies of this invention can be used therapeutically, such as tobind to and thereby neutralize or inhibit excess amounts of NAT in vivoafter administration. The antibodies can further be used for diagnosticpurposes, such as in labeled form to detect the presence of NAT in abody fluid, tissue sample or other extract, in accordance with knowndiagnostic methods.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The invention is further illustrated in the following examples.

EXAMPLE 1 Derivation of NAT Sequence

An effective way to produce fibrolase is to express it initially aspreprofibolase in which cleavage by the protease kex-2 occurs at thejunction of the “prepro” and “mature” regions to yield biologicallyactive material (“mature” fibrolase). From this design, the synthesisand processing of the preprofibrolase leads to secretion of maturefibrolase into the culture medium. The actual sequence at the cleavedjunction is ( . . . TKR↓QQRF . . . ).

Kex-2 is an endoprotease that cleaves after two adjacent basic aminoacids, in this case lysine(K)-arginine(R). Mature fibrolase expressedfrom DNA having the above mentioned sequence revealed that the expectedN-terminal glutamine (Q) residue had in fact undergone deamidation andcyclization to generate pyroglutamic acid (E). This chemicalmodification was deemed undesirable, since peptides with an N-terminalcyclized glutamine (pyroglutamic acid) residue fail to react in theEdman-degradation procedure for amino acid sequencing. Accordingly, bothof the glutamine (Q) residues at the N-terminus in the sequence formature fibrolase were deleted, resulting in an N-terminal arginine (R)residue. Since kex-2 cleaves after two adjacent basic amino acids asmentioned, it was anticipated that the sequence ( . . . KRRF . . . )would present an ambiguous site for kex-2 cleavage. Accordingly, theN-terminal arginine (R) residue (shown underlined above) was replacedwith a serine (S) residue to result in the sequence ( . . . KRSF . . .). The choice of serine was based on the need to introduce an amino acidwhich facilitates kex-2 cleavage when it occurs on the C-terminal sideof the hydrolysis site. Rholam et al., European Journal of Biochemistry,Volume 227, pages 707-714 (1995).

As a result, the DNA sequence for preprofibrolase was modified bysite-directed mutagenesis at the N-terminal coding region for maturefibrolase to substitute the codons for “QQR” with a codon for “S”, usinga standard PCR protocol, thus resulting in preproNAT having the aminoacid sequence of SEQ ID NO: 3. The oligonucleotides used to prime thePCR reactions are listed below, and their homology with the targetsequence is also shown. Initially, two PCR reactions were carried outusing oligos 1 and 4 as one primer pair and oligos 2 and 3 as anotherprimer pair, both with DNA of the parent gene as the template. The DNAproducts of these two reactions (601 and 815 nucleotides in length) werepurified by agarose gel electrophoresis and combined to serve as atemplate in a second round of PCR using oligos 1 and 2 as the primerpair. This final PCR product (1372 nucleotides in length) was cleavedwith restriction endonucleases XhoI and NotI. The digest wasdeproteinized with phenol/chloroform and DNA precipitated. A portion ofthe recovered DNA was ligated into the plasmid pPICZα (Invitrogen,Carlsbad, Calif., Catalog No. VI95-20), which had been similarly cleavedwith restriction endonucleases XhoI and NotI, enzymaticallydephosphorylated, and deproteinized with phenol/chloroform. Allsubsequent steps were carried out according to the Invitrogen PichiaExpression Kit manual (Invitrogen Corp., Catalog No. K1710-01). Theligation reaction products were transformed into E. coli byelectroporation and selected for survival on zeocin-containing solidmedia. The plasmid was isolated and the profibrolase region wasconfirmed by DNA sequencing. The plasmid was linearized by cleaving withrestriction endonuclease PmeI and then transformed into Pichia pastorisGS115his⁺. The GS115 strain is normally his⁻, so the his⁺ genotype wasrestored by transformation with a DNA source carrying the wild typeversion of the his4 gene. Alternatively, a his⁺ strain can be obtainedcommercially from Invitrogen Corp. (X-33 cell line, Catalog No.C180-00). Integrants were selected as zeocin-resistant colonies.Candidate clones were induced in methanol-containing media, and thebroth was assayed for NAT production on 4-20% PAGE, using Coomassiestaining.

The oligonucleotides used for site-directed PCR mutagenesis were asfollows: Oligo 1 (SEQ ID NO: 8) 5′-TACTATTGCCAGCATTGCTGC-3′ Oligo 2 (SEQID NO: 9) 5′-GCAAATGGCATTCTGACATCC-3′ Oligo 3 (SEQ ID NO: 10)5′-TCCAATTAAACTTGACTAAGAGATCTTTCCCACAAAGATACGTA C-3′ Oligo 4 (SEQ ID NO:11) 5′-GTACGTATCTTTGTGGGAAAGATCTCTTAGTCAAGTTTAATTGG-3′

The location of these oligonucleotides is shown below in relation to thedouble-stranded DNA sequence (SEQ ID NO: 12 coding or sense strand, SEQID NO: 13 complementary or antisense strand) and corresponding aminoacid sequence (SEQ ID NO: 14) of fibrolase (including the prepro region)being modified to create NAT. The N-terminal and C-terminal regions ofmature fibrolase are indicated by underlining of terminal amino acidsequences (QQRF and LNKP). The N-terminal region (QQRF) is the one beingmodified (to substitute S for QQR). For oligos 3 and 4, below, dashedlines are inserted to denote the location of the omitted codons encodingfor residues QQ in the N-terminal region of fibrolase.ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TACTCTAAAGGAAGTTAAAAATGACGACAAAATAAGCGTCGTAGGAGGCGTAATCGACGA M R F P S IF T A V L F A A S S A L A ACCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+GGTCAGTTGTGATGTTGTCTTCTACTTTGCCGTGTTTAAGGCCGACTTCGACAGTAGCCA P V N T T TE D E T A Q I P A E A V I GTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+ATGAGTCTAAATCTTCCCCTAAAGCTACAACGACAAAACGGTAAAAGGTTGTCGTGTTTA Y S D L E GD F D V A V L P F S N S T N             Oligo1      5′-TACTATTGCCAGCATTGCTGC-3′AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TTGCCCAATAACAAATATTTATGATGATAACGGTCGTAACGACGATTTCTTCTTCCCCAT N G L L F IN T T I A S I A A K E E G V XhoI    |TCTCTCGAGAAAAGAGAGGCTGAAGCTTCTTCTATTATCTTGGAATCTGGTAACGTTAAC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+AGAGAGCTCTTTTCTCTCCGACTTCGAAGAAGATAATAGAACCTTAGACCATTGCAATTG S L E K R EA E A S S I I L E S G N V NGATTACGAAGTTGTTTATCCAAGAAAGGTCACTCCAGTTCCTAGGGGTGCTGTTCAACCA−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+CTAATGCTTCAACAAATAGGTTCTTTCCAGTGAGGTCAAGGATCCCCACGACAAGTTGGT D Y E V V YP R K V T P V P R G A V Q PAAGTACGAAGATGCCATGCAATACGAATTCAAGGTTAACAGTGAACCAGTTGTCTTGCAC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TTCATGCTTCTACGGTACGTTATGCTTAAGTTCCAATTGTCACTTGGTCAACAGAACGTG K Y E D A MQ Y E F K V N S E P V V L HTTGGAAAAAAACAAAGGTTTGTTCTCTGAAGATTACTCTGAAACTCATTACTCCCCAGAT−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+AACCTTTTTTTGTTTCCAAACAAGAGACTTCTAATGAGACTTTGAGTAATGAGGGGTCTA L E K N K GL F S E D Y S E T H Y S P DGGTAGAGAAATTACTACTTACCCATTGGGTGAAGATCACTGTTACTACCATGGTAGAATC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+CCATCTCTTTAATGATGAATGGGTAACCCACTTCTAGTGACAATGATGGTACCATCTTAG G R E I T TY P L G E D H C Y Y H G R IGAAAACGATGCTGACTCCACTGCTTCTATCTCTGCTTGTAACGGTTTGAAGGGTCATTTC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+CTTTTGCTACGACTGAGGTGACGAAGATAGAGACGAACATTGCCAAACTTCCCAGTAAAG E N D A D ST A S I S A C N G L K G H FAAGTTGCAAGGTGAAATGTACTTGATTGAACCATTGGAATTGTCCGACTCTGAAGCCCAT−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TTCAACGTTCCACTTTACATGAACTAACTTGGTAACCTTAACAGGCTGAGACTTCGGGTA K L Q G E MY L I E P L E L S D S E A HGCTGTCTACAAGTACGAAAACGTCGAAAAGGAAGATGAAGCCCCAAAGATGTGTGGTGTT−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+CGACAGATGTTCATGCTTTTGCAGCTTTTCCTTCTACTTCGGGGTTTCTACACACCACAA A V Y K Y EN V E K E D E A P K M C G V                                Oligo3′ 5′-TCCAATTAAACTTGACTAAGACCCAAAACTGGGAATCATATGAACCAATCAAGAAGGCCTTCCAATTAAACTTGACTAAG−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TGGGTTTTGACCCTTAGTATACTTGGTTAGTTCTTCCGGAAGGTTAATTTGAACTGATTC                    Oligo 4   3′-GGTTAATTTGAACTGATTC T Q N W E S Y E P IK K A F Q L N L T K AGA−−−−−−TCTTTCCCACAAAGATACGTAC-3′AGACAACAAAGATTCCCACAAAGATACGTACAGCTGGTTATCGTTGCTGACCACCGTATG−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TCTGTTGTTTCTAAGGGTGTTTCTATGCATGTCGACCAATAGCAACGACTGGTGGCATACTCT------AGAAAGGGTGTTTCTATGCATG-5′ P Q Q R F P Q R Y V Q L V I V A D H RM AACACTAAATACAACGGTGACTCTGACAAAATCCGTCAATGGGTGCACCAAATCGTCAAC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TTGTGATTTATGTTGCCACTGAGACTGTTTTAGGCAGTTACCCACGTGGTTTAGCAGTTG N T K Y N GD S D K I R Q W V H Q I V NACCATTAACGAAATCTACAGACCACTGAACATCCAATTCACTTTGGTTGGTTTGGAAATC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TGGTAATTGCTTTAGATGTCTGGTGACTTGTAGGTTAAGTGAAACCAACCAAACCTTTAG T I N E I YR P L N I Q F T L V G L E ITGGTCCAACCAAGATTTGATCACCGTTACTTCTGTATCCCACGACACTCTGGCATCCTTC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+ACCAGGTTGGTTCTAAACTAGTGGCAATGAAGACATAGGGTGCTGTGAGACCGTAGGAAG W S N Q D LI T V T S V S H D T L A S FGGTAACTGGCGTGAAACCGACCTGCTGCGTCGCCAACGTCATGATAACGCTCAACTGCTG−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+CCATTGACCGCACTTTGGCTGGACGACGCAGCGGTTGCAGTACTATTGCGAGTTGACGAC G N W R E TD L L R R Q R H D N A Q L LACCGCTATCGACTTCGACGGTGATACTGTTGGTCTGGCTTACGTTGGTGGCATGTGTCAA−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TGGCGATAGCTGAAGCTGCCACTATGACAACCAGACCGAATGCAACCACCGTACACAGTT T A I D F DG D T V G L A Y V G G M C QCTGAAACATTCTACTGGTGTTATCCAGGACCACTCCGCTATTAACCTGCTGGTTGCTCTG−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+GACTTTGTAAGATGACCACAATAGGTCCTGGTGAGGCGATAATTGGACGACCAACGAGAC L K H S T GV I Q D H S A I N L L V A LACCATGGCACACGAACTGGGTCATAACCTGGGTATGAACCACGATGGCAACCAGTGTCAC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TGGTACCGTGTGCTTGACCCAGTATTGGACCCATACTTGGTGCTACCGTTGGTCACAGTG T M A H E LG H N L G M N H D G N Q C HTGCGGTGCAAACTCCTGTGTTATGGCTGCTATGCTGTCCGATCAACCATCCAAACTGTTC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+ACGCCACGTTTGAGGACACAATACCGACGATACGACAGGCTAGTTGGTAGGTTTGACAAG C G A N S CV M A A M L S D Q P S K L FTCCGACTGCTCTAAGAAAGACTACCAGACCTTCCTGACCGTTAACAACCCGCAGTGTATC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+AGGCTGACGAGATTCTTTCTGATGGTCTGGAAGGACTGGCAATTGTTGGGCGTCACATAG S D C S K KD Y Q T F L T V N N P Q C I              NotI                 |CTGAACAAACCGTAAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAAGAGGAT−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+GACTTGTTTGGCATTCGCCGGCGGTCGAAAGATCTTGTTTTTGAGTAGAGTCTTCTCCTA L N K P * AA A S F L E Q K L I S E E DCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTGTAGCCTTAGACATGAC−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+GACTTATCGCGGCAGCTGGTAGTAGTAGTAGTAGTAACTCAAACATCGGAATCTGTACTG L N S A V DH H H H H H * V C S L R H DTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAG−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+ACAAGGAGTCAAGTTCAACCCGTGAATGCTCTTCTGGCCAGAACGATCTAAGATTAGTTC C S S V Q VG H L R E D R S C * I L I KAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATT−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+TCCTACAGTCTTACGGTAAACGGACTCTCTACGTCCGAAGTAAAAACTATGAAAAAATAA CCTACAGTCTTACGGTAAACG-5′ Oligo 2 R M S E C H L P E R C R L H F * Y F FI

EXAMPLE 2 Expression of NAT in Pichia pastoris

When attempts were made to express the DNA for NAT in E. coli, very poorrefolding and a requirement for dilute conditions reduced thepurification efficiency. These and other considerations led to the usageof Pichia pastoris, a yeast species, as the host cell. A culture ofselected clones of Pichia pastoris which had been transfected withprepro NAT cDNA (SEQ ID NO: 4) was inoculated into 500 ml of thefollowing inoculation growth medium: Per liter of batch medium Yeastextract 30.0 g Potassium phosphate dibasic 17.2 g Glucose 20.0 g Biotin0.004 g Water to 1 liter Phosphoric acid, 85% to adjust pH to 6.00

The transfected P. pastoris cells were incubated at 30° C. in a shakerfor about 30 to 32 hours. About 1% (w/v) of the resulting culture wasused to inoculate a 10-liter fermentor. The fermentor containedsterilized basal salts and glucose (below). Twelve milliliters per literof PTM4 salts (PTM4 is a trace metals solution containing cupric sulfatepentahydrate, sodium iodide, manganese sulfate monohydrate, sodiummolybdate dihydrate, boric acid, cobaltous chloride hexahydrate, zincchloride, ferrous sulfate heptahydrate, d-biotin, sulfuric acid andpurified water) were added per liter of batch medium after fermentorsterilization. The fermentation growth temperature was 30° C. Thefermentor pH was controlled with ammonium hydroxide and phosphoric acidat pH 6.00. Zinc from the zinc salts added to the medium becomesincorporated into NAT as part of the metalloproteinase structure. Basalsalts per liter of batch medium Phosphoric acid, 85% 26.7 ml Calciumsulfate 0.93 g Potassium sulfate 18.2 g Magnesium sulfate-7H₂O 14.9 gPotassium hydroxide 4.13 g Glucose 30.0 g Water to 1 liter

The batch culture was grown until the glucose was completely consumed(17 to 20 hours). Then a fed-batch phase was initiated. The fed-batchphase media consisted of glucose and 12 ml of PTM4 salts per liter. Theinduction feed consisted of glucose, 25% methanol, and 12 ml of PTM4salts per liter. At induction, the temperature of the reactor wasshifted to 20° C. The induction phase lasted 60 to 75 hours. Theconditioned media were harvested and the cellular debris was discarded.

EXAMPLE 3 Purification of NAT from Pichia pastoris

The yeast broth (conditioned media less cellular debris) from Example 2was clarified and the pH and conductivity were adjusted to 6.5 and 10-20mS/cm, respectively. The broth was loaded onto an immobilized metalaffinity resin that had been charged with copper (Cu) and equilibratedwith phosphate buffered saline (PBS). The resin was washed with PBS andeluted with an imidazole gradient (0-100 mM) in PBS. Fractionscontaining “mature” NAT (SEQ ID NO: 1) were pooled and diluted until theconductivity was less than 1.5 mS/cm, pH 6.4. The diluted pool wasloaded onto an SP Sepharose resin (Amersham Pharmacia Biotech, Inc.,Piscataway, N.J.) that had been equilibrated with 10 mM2-(N-morpholino)ethanesulfonic acid (MES). The column was washed withMES and eluted with a NaCl gradient (0-500 mM) in MES. Fractionscontaining NAT were pooled and stored.

EXAMPLE 4

Thrombolysis in Acute Thrombosis of Rat Carotid Artery; Comparison ofNAT with Urokinase

To demonstrate that “mature” NAT (SEQ ID NO: 1) is biologically activeand functionally unique, acute pharmacology studies were conducted inrats where focal injury to one of the carotid arteries was created byapplying anodal current. This injury produces an occlusive thrombuswhich generally forms within fifteen minutes. Once the thrombus wasformed, the artery was observed for a period of thirty minutes to assurethat the carotid occlusion was stable. Then heparin and aspirin wereadministered intravenously to prevent further propagation of thethrombus. The animals were then treated with an intraarterial infusionof test material. Blood flow through the carotid artery was monitoredduring the delivery of test material so that successful clot lysis couldbe detected and the time at which clot lysis occurred could be noted.The percentage of experiments where clot lysis occurred was noted andgroup means were calculated for only those experiments where clot lysiswas successful. As a measure of the hemorrhagic potential of the testmaterial, any blood that was shed from the surgical site was collectedwith gauze swabs. The swabs were placed in a detergent solution tosolubilize red blood cells and release hemoglobin, which was thenquantified spectrophotometrically. Shed hemoglobin was used to calculatea volume of blood loss. Test data are reported in the Table below. TABLE1 Incidence of Clot Lysis, Time to Clot Lysis and Surgical Blood Loss(Mean ± std. dev.) INCIDENCE TIME TO OF LYSIS LYSIS BLOOD LOSS (%) (min)(ml) SALINE  0% N/A 0.10 ± 0.23 (n = 6) (0 of 6) Urokinase 33% 55.3 ±15.9 1.06 ± 1.59 25 U/min  (5 of 15) (n = 15) Urokinase 86% 33.5 ± 15.31.43 ± 1.45 250 U/min (13 of 15) (n = 15) NAT 2 mg 78% 6.3 ± 5.8 0.96 ±0.77 (n = 14) (11 of 14)

These studies establish that NAT is biologically active in an animalmodel of in vivo clot lysis. Further, clot lysis was achieved in amarkedly reduced amount of time and with less blood loss from thesurgical site, in comparison with urokinase. Thus, the activity profileof NAT can be distinguished from the plasminogen activator class ofthrombolytic agents (represented by urokinase) in that clot lysis withNAT occurs more rapidly and with reduced hemorrhagic complications.

The fibrinolytic activity of NAT is comparable to that of fibrolase. Inaddition, as mentioned above the stability of the N-terminus of NATresults in a more homogeneous end product upon recombinant expression,which is a distinct advantage (i.e., the N-terminus will not change overtime resulting in a mixture of different forms, thus making thepolypeptide more stable).

1. A thrombolytically active variant of SEQ ID NO:5, wherein thesequence of amino acid residues Gln-Gln-Arg at positions 1-3 of SEQ IDNO:5 is substituted with an amino acid that facilitates kex-2 cleavagewhen the amino acid occurs on the C-terminal side of the kex-2hydrolysis site, and the remainder of the amino acid sequence of SEQ IDNO:5 is unchanged.
 2. A fusion polypeptide comprising thethrombolytically active variant of claim 1 fused to a heterologouspolypeptide sequence.
 3. A composition comprising the thrombolyticallyactive variant of claim 1, and one or more components selected from apharmaceutically acceptable diluent, a pharmaceutically acceptablepreservative, a pharmaceutically acceptable solubilizer, apharmaceutically acceptable emulsifier, a pharmaceutically acceptableadjuvant, or a pharmaceutically acceptable carrier.
 4. A method fortreating thrombosis in a mammal comprising administering locally to aclot in a blood vessel of the mammal a thrombolytically effective amountof the composition of claim
 3. 5. The method of claim 4, wherein thecomposition is administered as two or more doses.
 6. The method of claim4, wherein the mammal is a human.
 7. A method for lysing a blood clotcomprising contacting the blood clot with a thrombolytically effectiveamount of the composition of claim
 3. 8. The method of claim 7, whereinthe blood clot is contacted in vivo.
 9. The method of claim 8, whereinthe blood clot is contacted in vivo in a human.
 10. The method of claim7, wherein the blood clot is contacted in vitro.
 11. A nucleic acidmolecule comprising a coding sequence encoding the thrombolyticallyactive variant of claim
 1. 12. An expression vector comprising thenucleic acid molecule of claim 11, operatively linked to expressionregulatory elements.
 13. A host cell comprising the expression vector ofclaim
 12. 14. The host cell of claim 13, wherein the cell is a yeastcell.
 15. The host cell of claim 14, wherein the yeast is Pichiapastoris.
 16. A method for producing a recombinant polypeptidecomprising providing a population of host cells according to claim 13and causing expression of the polypeptide.
 17. A method for producing arecombinant polypeptide comprising providing a population of host cellsaccording to claim 14 and causing expression of the polypeptide.
 18. Amethod for producing a recombinant polypeptide comprising providing apopulation of host cells according to claim 15 and causing expression ofthe polypeptide.